Gaining detailed knowledge on the workings of the vesicular transmitter uptake machinery is important in understanding synaptic function and plasticity, because this uptake process governs directly the number of transmitters released into the synaptic cleft and thus the degree of activation of the postsynaptic receptors. Determining the mechanism and rates of vesicular transmitter uptake also is important towards understanding the kinetics of vesicle recycling at the synapse and the energy requirements for loading and maintaining the filled vesicle at the presynaptic terminal. Given synaptic transmission is dependent on the uptake and storage of neurotransmitters within vesicles, it is not surprising that they are sites of action for many drugs, and thus understanding the functioning of this important molecular machinery will also have important pharmacological implications. Our proposed single-molecule and single-vesicle experiments are targeted towards deciphering the molecular organization and mechanism by which neurotransmitters are loaded into the synaptic vesicle. Towards this end, our specific aims are: Aim 1: Rotational measurements of synaptic vesicle integral membrane proteins and of small molecules contained within the vesicle Aim 2: Single-molecule studies of the vesicular H+ATPase Aim 3: Single-molecule studies of the vesicular glutamate transporters Aim 4: Investigation of the coupling of transmitter uptake to the glycolytic enzymes on synaptic vesicles and the spatial organization of the uptake machinery. From these experiments, we will develop an in-depth understanding of the workings of this complex molecular machinery with single-molecule resolutions, and offer new insight into how malfunctioning caused by neurological diseases or targeted disruption of this machinery with drugs can affect synaptic transmission. Project Narrative: To orchestrate neurotransmission, more than one thousand proteins are present at the presynaptic terminal and which either directly or indirectly interact with the synaptic vesicle, with the end result being the regulated release of neurotransmitters into the synaptic cleft. This high degree of convergence of presynaptic functions onto the synaptic vesicle has led to a "vesicocentric" view of neurotransmission that focuses on the synaptic vesicle as the central organelle in synaptic function. This proposal describes plans to study the synaptic vesicle with single-molecule resolutions, because discerning the detailed workings of this important organelle is critical in achieving the next level of quantitative understanding of synaptic transmission and malfunctioning caused by neurological diseases.